# Preparation of ESAT6-Fc Fusion Protein and Its Therapeutic Efficacy and Immune Mechanisms in Allergic Asthma Mice via Intranasal Immunization

## TL;DR

This study shows that an ESAT6-Fc fusion protein, delivered intranasally, can reduce allergic asthma symptoms in mice by shifting immune responses toward a healthier balance.

## Contribution

The novel contribution is the development and evaluation of an ESAT6-Fc fusion protein as a potential intranasal immunotherapy for allergic asthma.

## Key findings

- ESAT6-Fc reduced airway inflammation and mucus production in allergic asthma mice.
- The fusion protein shifted immune cell proportions by increasing Th1 cells and decreasing Th2 and Th17 cells.
- CD3e and CD3g were identified as key factors in the immune regulatory mechanism of ESAT6-Fc.

## Abstract

The respiratory mucosal system plays a critical role in the pathogenesis of allergic asthma (AA). Currently, therapeutic Fc fusion proteins are as a promising strategy for mucosal vaccine delivery systems. In this work, a plasmid encoding the Mycobacterium tuberculosis ESAT6-Fc fusion protein was successfully constructed, and high-purity ESAT6-Fc fusion protein was subsequently obtained. Administered via intranasal immunization in OVA-induced allergic asthma model mice, ESAT6-Fc fusion protein significantly alleviated airway inflammation and mucus production, and reduced the proportions of Th2 cells, Th17 cells, and eosinophils, while increasing the proportions of Th1 cells with no histopathological changes to major organs. To elucidate the underlying immune regulatory mechanisms of ESAT6, integrated transcriptomic and proteomic analyses were performed, revealing Th1/Th2 cell differentiation and Th17 cell differentiation as the two most significantly enriched pathways at both the gene and protein levels. CD3e (CD3E) and CD3g (CD3G), two essential subunits of the TCR–CD3 complex, were identified as core target factors. The validations from the ESAT6-Fc-treated AA lung tissues, as well as co-cultured TH0 cells from C57BL/6J mice and CD2.4 dendritic cells exposed to the ESAT6-Fc protein, were consistent with the aforementioned findings. ESAT6-Fc exhibits a safe profile with favorable efficacy against OVA-induced AA via intranasal immunization, and ESAT6 ameliorates AA by regulating the differentiation of Th0 cells into Th1 cells, which were closely associated with the down-regulation of CD3e and CD3g expression, presumably leading to the impairment of TCR–CD3 complex assembly. ESAT6-Fc fusion protein demonstrates promise as a potential safe intranasal immunotherapy agent for the treatment of AA.